Otto C M, Niagro F, Su X, Rawlings C A
College of Veterinary Medicine, Department of Small Animal Medicine, University of Georgia, Athens, USA.
Clin Diagn Lab Immunol. 1995 Nov;2(6):740-6. doi: 10.1128/cdli.2.6.740-746.1995.
The tumor necrosis factor (TNF) genes from cats, horses, and pigs have all been cloned into the pFLAG-1 fusion protein expression vector (International Biotechnologies, Inc., Kodak, New Haven, Conn.). Growth curves for Escherichia coli containing the pFLAG-1 vector alone and the pFLAG-1 vector containing the TNF gene from each species were determined by visible light spectrophotometry (at 600 nm). Porcine TNF, equine TNF, and feline TNF cultures had slower doubling rates than cultures containing the pFLAG-1 vector alone. Cultures of cells transformed with feline TNF reached peak densities at 3 to 4 h and then decreased to near initial densities prior to the recovery of growth. The induction of expression with isopropyl-beta-D-thiogalactopyranoside (IPTG) arrested the growth of fresh feline TNF cultures for 6 h, which was followed by complete recovery. This inhibition occurred in two strains of E. coli (LL308 and JM101). Induced feline TNF cultures expressed the TNF-FLAG fusion protein for the first 6.5 h. Uninduced cultures expressed low levels of fusion protein. The feline TNF-pFLAG-1 vector was purified from cells expressing fusion protein and from cells with recovered growth curves. Sequencing the vector demonstrated the complete feline TNF gene and tac promoter in cells expressing the fusion protein and a deletional mutation of the tac promoter site in recovered cells. In contrast to equine and porcine TNF, the expression of recombinant feline TNF is toxic to E. coli. Alterations in protein folding and the prevention of secretion of the feline protein may explain the toxic effect.
猫、马和猪的肿瘤坏死因子（TNF）基因均已被克隆到pFLAG-1融合蛋白表达载体中（国际生物技术公司，柯达，康涅狄格州纽黑文）。通过可见光分光光度法（在600nm处）测定单独含有pFLAG-1载体以及含有各物种TNF基因的pFLAG-1载体的大肠杆菌的生长曲线。与单独含有pFLAG-1载体的培养物相比，猪TNF、马TNF和猫TNF培养物的倍增速率较慢。用猫TNF转化的细胞培养物在3至4小时达到峰值密度，然后在恢复生长之前降至接近初始密度。用异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达使新鲜的猫TNF培养物的生长停滞6小时，随后完全恢复。这种抑制作用发生在两株大肠杆菌（LL308和JM101）中。诱导的猫TNF培养物在最初的6.5小时表达TNF-FLAG融合蛋白。未诱导的培养物表达低水平的融合蛋白。从表达融合蛋白的细胞以及具有恢复生长曲线的细胞中纯化猫TNF-pFLAG-1载体。对载体进行测序表明，在表达融合蛋白的细胞中存在完整的猫TNF基因和tac启动子，而在恢复的细胞中tac启动子位点存在缺失突变。与马和猪TNF不同，重组猫TNF的表达对大肠杆菌有毒性。蛋白质折叠的改变以及猫蛋白分泌的阻止可能解释了这种毒性作用。
